Cancer is a major disease that threatens human lives. In South Korea, cancer has been No. 1 cause of death for the past several years. Cancer is the second major cause of death in the U.S.A following the cardiovascular diseases. Although numerous researches have been and are currently conducted, cancer is still the biggest catastrophe that human race has ever suffered, and it takes millions of lives and astronomical costs every year.
Cancer can be referred to as genetic disease in cellular level, considering that the disease develops as genes develop mutations such as oncogenes and tumor suppressor genes. Currently available cancer treatment includes surgery, chemotherapy, radiation therapy and immunotherapy, but the issues related to inhibition of malignant tumor and recurrence thereof have not had efficient solution yet.
One of the most important biological traits of cancer is that the cancer can migrate, and this proposes the biggest obstacle to finding the cure for cancer. In effect, approximately 60% of all the patients with solid tumor showed minute, but clinically-migrated tumor in the diagnosis of primary tumor, and it has been widely recognized that the most critical cause of death of most cancer patients is the metastasis. The process of metastasis involves penetration of tumor to local tissue along with formation of new vessels (i.e., angiogenesis) in which tumor angiogenesis factor (TAF) involves. The vessels newly generated by the tumor have many deficiencies, allowing the cancer cells to easily penetrate. Cancer penetration and metastasis require numerous receptors on cancer cell surfaces such as laminin receptor which is necessary for the adherence to matrix and basement membrane of the tissue, various enzymes necessary to dissolve stroma of normal tissues such as collagenase Type IV, plasminogen activator and cathepsin D, growth factor, autocrine motility factor (AMF), and expression of oncogenes.
Huge expectations are laid on the substances with inhibitory effect on metastasis, but few have actually been developed with an aim to inhibit metastasis. Currently, substances including sulfated polysaccharide, N-diazo acetyl glycin derivative, neuraminidase and fibronectins (FNs) enzyme have been reported of metastasis inhibition effect. But none of these was reported to have been commercialized, and it has not been clarified yet that said substances themselves have such metastasis inhibition effect. If a method for inhibiting migration of cancer efficiently is developed, a treatment that can effectively suppress the deaths by metastasis will be made available.
Meanwhile, delta-like 1 homolog (DLK1), a member of notch/delta/serrate family, is transmembrane glycoprotein encoded in dlk1 located at gene 14q32, and consists of 383 amino acids containing 280 extracellular regions, 24 transmembrane segmemts, and 56 cytoplasmic domains. Among these, there are 6 epidermal growth factor (EGF)-like repeat domains on extracellular region, having 3 N-glycosylation and 7 0-glycosylation sites. As explained, DLK1 is transmembrane protein, but it is also known to be the protein that is an extracellular portion shed from the cellular membrane due to tumor necrosis factor alpha, converting enzyme (TACE) and has a separate function (Yuhui Wang and Hei Sook Sul, Molecular and cellularbiology. 26(14): 5421-5435, 2006).
DLK1 is found in various forms of 50˜60 kDa by the glycosylation on the cellular membrane (Smas C M and Sul H S, Cell. 73:725-34, 1993), and has 4 splicing variants by the alternative splicing (Smas C M et al., Biochemistry. 33:9257-65, 1994). Among these, two larger variants have cleavage sites of proteolytic enzymes which are cut by TACE to generate two soluble forms sized to 50 kDa and 25 kDa (Yuhui Wang et al., Journal of Nutrition. 136:2953-2956, 2006) (See FIG. 1).
DLK1 is widely known as fetal antigen 1 (FA1) (Jensen C H et al., European Journal of Biochemistry. 225:83-92, 1994), since this is expressed mainly in developmental stage from embryonic tissue (Smas C M et al., Cell. 73:725-34, 1993; Kaneta M et al., Journal of Immunology. 164:256-64, 2000) and placenta, and particularly in high concentration from maternal serum. Some report expression of DLK1 in glandular cell of pancreas (Kaneta M et al., Journal of Immunology. 164:256-64, 2000), ovary cell, or skeletal myotubes (Floridon C et al., Differentiation. 66:49-59, 2000). DLK1 expression disappears from most tissues after birth, and appears at limited cells such as preadipocyte (Smas C M et al., Cell. 73:725-34, 1993), pancreatic islet cell (Carlsson C at al., Endocrinology. 138:3940-8, 1997) thymic stromal cell (Kaneta M et al., Journal of Immunology. 164:256-64, 2000), or adrenal gland cell (Halder S K et al., Endocrinology. 139:3316-28, 1998). DLK1 expression is also known as paternal manoallelic expression due to influence by methylation (Schmidt J V at al., Genes and Development. 14:1997-2002, 2000; Takada S et al., Current Biology 10:1135-8, 2000; Wylic A A at al, Genome Research. 10:1711-8, 2000).
DLK1 is widely known as preadipocyte factor-1 (Pref-1) that plays a role of inhibiting differentiation of adipocyte and most frequently researched in that regard (Smas C M et al., Cell. 73:725-34; Villena J A et al., Hormone and Metabolic Research. 34:664-70, 2002). Beside the inhibition of the adipocyte differentiation, DLK1 is also known as it inhibits differentiation of hematopoietic stem cells (Sakajiri S et al., Leukemia. 19:1404-10, 2005; Li L et al., Oncogene. 24:4472-6, 2005), regulates differentiation of lymphoid progenitor cell (Bauer S R et al., Molecular and Cellular Biology. 18:5247-55, 1998; Kaneta M et al., Journal of Immunology. 164:256-64, 2000) and is involved in wound healing (Samulewicz S J et al., Wound Repair and Regeneration. 10:215-21, 2002). However, a little has been studied on the role of DLK1 in relation to cancer cells.
Studies on a link between DLK1 and a few types of cancers has recently reported over-expression of DLK1 in glioma, and the finding that cDNA of DLK1, if over-expressed in glioma, increased proliferation of glioma and thus increased migration (Yin D et al., Oncogene. 25:1852-61, 2006). The report also indicated that DLK1 expression in liver cancer is increased compared to that in normal liver cells, and that by siRNA test, the tumor greatly shrinks when DLK1 expression is decreased (Huang J et al., Carcinogenesis. 28(5):1094-1103, 2007). It has been reported recently that the cytoplasmic domain of DLK1 plays an important role in the tumorigenesis (Yuri K et al., Cancer Research. 69(24):OF1-10, 2009). Until now, studies about soluble DLK1, which is the extracellular portion shed from the cellular membrane by TACE, have been mainly focused on the function of inhibiting differentiation of adipocyte. The linkage between the extracellular soluble domain of DLK1 and cancer has not been studied yet.
Therefore, the inventors completed the present invention by establishing recombinant expression vector comprising the soluble domain gene in extracellular region of DLK1 with genes of Fc domain of IgG antigen, expressing and purifying DLK1-Fc fusion protein from 293E cell, and confirming markedly decreased migration of cancer cell by DLK1-Fc fusion protein, and efficacy as a drug to inhibit metastasis through measurement of pharmacokinetic (PK) parameters, thereby confirming that the DLK1-Fc fusion protein can be efficaciously used as an effective ingredient of compositions for inhibiting metastasis.